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Promoter methylation rates of three tumor suppressor genes were detected in the total cirDNA, derived from plasma and cell-surface-bound fractions, of gastric cancer patients (n = 30) and healthy subjects (n = 30) using MS-PCR. The tumor marker protein levels (CEA, CA 72.4, CA 19.9) were assessed in plasma samples by commercial immunoassay kits. Methylated forms of p15, MGMT and hMLH1genes were detected in the total cirDNA with high rates at II, III and IV stages of gastric cancer. Sensitivity of the MS-PCR-based assay for at least one of methylated p15 and hMLH1 genes in gastric cancer was found to be much higher compared with the sensitivity of the immunoassay for elevated levels of CA 72.4 and CA 19.9 proteins (63 and 30%, respectively). No significant correlation was found between epigenetic and protein markers so indicating their independent development in gastric tumor pathogenesis. To conclude, epigenetic alterations of total cirDNA and elevated levels of tumor-associated proteins were found to be independently associated with gastric cancer indicating their usefulness as complementary diagnostic and prognostic markers for gastric cancer.

The concentration of cell-free DNA and promoter methylation status of the MGMT, p15, and hMLH1 genes were analyzed by a fluorescence-based assay and methylation-specific PCR (MSP) in the blood of gastric cancer patients (n= 20) and healthy subjects (n= 22). Gastric cancer patients were characterized by an increased concentration of circulating DNA in the plasma; the amount of cell-surface-bound DNA was not decreased compared with controls and amounted to 80 +/- 15% of the total circulating DNA. MSP analysis of three genes in the cell-surface-bound DNA permits the detection of gastric cancer patients with a sensitivity of 75% and a specificity of 54%. Thus, the cell-surface-bound DNA is a convenient source of DNA for MSP analysis of cancer-specific markers. The data on the presence of methylated DNA in plasma combined with the analysis of other cancer-related changes in DNA can significantly contribute to cancer diagnostics.